Epstein-Barr virus uses different complexes of glycoproteins gH and gL to infect B lymphocytes and epithelial cells

The Epstein-Barr virus (EBV) gH-gL complex includes a third glycoprotein, gp42. gp42 binds to HLA class II on the surfaces of B lymphocytes, and this interaction is essential for infection of the B cell. We report here that, in contrast, gp42 is dispensable for infection of epithelial cell line SVKCR2. A soluble form of gp42, gp42.Fc, can, however, inhibit infection of both cell types. Soluble gp42 can interact with EBV gH and gL and can rescue the ability of virus lacking gp42 to transform B cells, suggesting that a gH-gL-gp42.Fc complex can be formed by extrinsic addition of the soluble protein. Truncated forms of gp42.Fc that retain the ability to bind HLA class II but that cannot interact with gH and gL still inhibit B-cell infection by wild-type virus but cannot inhibit infection of SVKCR2 cells or rescue the ability of recombinant gp42-negative virus to transform B cells. An analysis of wild-type virions indicates the presence of more gH and gL than gp42. To explain these results, we describe a model in which wild-type EBV virions are proposed to contain two types of gH-gL complexes, one that includes gp42 and one that does not. We further propose that these two forms of the complex have mutually exclusive abilities to mediate the infection of B cells and epithelial cells. Conversion of one to the other concurrently alters the ability of virus to infect each cell type. The model also suggests that epithelial cells may express a molecule that serves the same cofactor function for this cell type as HLA class II does for B cells and that the gH-gL complex interacts directly with this putative epithelial cofactor.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes

Epstein-Barr virus (EBV) infection is associated with several human diseases that involve unrestricted proliferation of B lymphocytes. EBV nuclear antigen 1 (EBNA-1) is expressed in all EBV-infected cells and plays an essential role in persistence of the EBV genome. EBNA-1 has also been reported to have oncogenic potential. As an approach for treating EBV infections, we examined the capacity of EBNA-1 ribozymes delivered by recombinant adenoviruses to suppress EBNA-1 expression and to block virus-induced B cell proliferation. In contrast to primary B cells, EBV-transformed B lymphoblastoid cell lines expressed alphav integrins, the adenovirus internalization receptors, and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of a specific ribozyme (RZ1) to lymphoblastoid cell lines, suppressed EBNA-1 mRNA and protein expression, significantly reduced the number of EBV genomes, and nearly abolished cell proliferation in low serum. Adenovirus delivery of RZ1 also prevented EBV infection of an established EBV-negative B cell line. These studies demonstrate the potential use of adenovirus-encoded ribozymes to treat EBV-induced lymphoproliferative disorders.     

Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.     

CD21-Dependent infection of an epithelial cell line, 293, by Epstein-Barr virus

Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21, that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1, was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers that have arisen to allow for elevated expression of LMP-1.     

A mechanism of T cell regulation of Epstein-Barr virus latency

The tumorigenic potential of B lymphocytes latently infected with EBV is effectively controlled by T cell immunity. The mechanisms of this T cell regulation, however, are incompletely understood. In this study, T lymphocytes were found to proliferate in response to serum-free supernatants of EBV-immortalized cells and to deplete them of growth factors required by the immortalized B cells for autocrine growth. Lactic acid was reported to account for approximately 90% of the autocrine growth factor activity in serum-free supernatants of EBV-immortalized cell lines. Synthetic lactic acid was now found to promote growth in activated T cells. In addition, B cell suppression resulting from coculture of EBV-infected B cells with autologous T cells was reversed by the addition of supernatants from EBV-immortalized cell lines. Thus, T cell competition for growth factors produced and utilized by EBV-immortalized B cells for continuous proliferation may represent an important and novel regulatory mechanism for the maintenance of EBV latency in B lymphocytes.     

Malignant histiocytosis derived from a common histiocyte clone in a patient with chronic Epstein-Barr virus infection

Epstein-Barr virus (EBV) has a particular propensity for B lymphocytes, but in a few cases it seems to play a role in histiocytic disorders and EVB DNA has been identified in histiocytes. To determine what kind of cell proliferate clonally, we studied a patient with malignant histiocytosis that developed after chronic EBV infection. Polymerase chain reaction (PCR) for lymphocyte-defined membrane antigen (LYDMA) of EBV, a marker of monoclonality, double stainings of cell markers (B, T lymphocytes; histiocytes), and in situ hybridization for EBV were performed in tissues obtained in 1987 and 1990 before the appearance of malignant histiocytosis and in 1991 after the disease was diagnosed. PCR for LYDMA from multiple samples during the disease showed the same single band, indicating that chronic EBV infection and malignant histiocytosis were caused by the same single virion. We also found a single terminal repeat band of EBV which supports this finding. In the studies of double stainings, EBV was present in histiocytes of the non-neoplastic early stage, and in the neoplastic cells of malignant histiocytosis. The histiocyte, infected with EBV, clonally expanded to result in malignant histiocytosis.     

Infection of leukaemic B lymphocytes by Epstein Barr virus

Epstein-Barr virus (EBV) infection of B lymphocytes in vitro gives rise to immortalized lymphoblastoid cell lines. Previous reports have shown that chronic lymphocytic leukaemia (CLL) cells, although infectable by EBV, are resistant to immortalization (1-4), although a small number of CLL cell lines have been reported (5-7). In the present study we have analysed early events occurring after EBV infection in 16 CLL samples. Out of 16 samples, 15 could be infected by the virus and expressed the full EB viral nuclear antigen (EBNA) complex but only one out of 16 expressed the latent membrane protein (LMP). The five CLLs in which we could investigate the presence of viral episomes showed circularized EBV by 16 hours after infection. The sequence of EBNA expression and genome circularization mirrored that seen in normal B cells, although genome amplification was not detected. The only CLL sample which expressed LMP after EBV infection was induced to proliferate for 2-3 weeks, but no cell line was established. Immortalized cell lines were obtained from three out of 16 samples tested, but all were polyclonal for light chain expression and had arisen from the CD5-negative, normal B-cell population. Thus the inability of EBV to induce proliferation of most CLL cells correlated with the absence of LMP expression which is invariably expressed during immortalization of normal B cells. This novel type of restricted gene expression could be compatible with evasion of host immune responses and consequent long-term survival of the cell in vivo.     

Epstein-Barr virus promotes epithelial cell growth in the absence of EBNA2 and LMP1 expression

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10(6) cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.     

An Epstein-Barr virus-associated superantigen

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.     

Preliminary evidence for an association of Epstein-Barr virus with pre-ulcerative oral lesions in patients with recurrent aphthous ulcers or Beh?et's disease

In this study we used the polymerase chain reaction (PCR), slot blot and Southern blot hybridization, direct sequencing and in situ hybridization (ISH) to show the possible presence of EBV-DNA in pre-ulcerative oral aphthous lesions of patients with recurrent aphthous ulcers (RAU) or Beh?et's disease (BD). For this purpose, formalin-fixed biopsy specimens were obtained from 13 pre-ulcerative oral aphthous lesions of nine RAU and four BD patients. Five specimens of normal oral mucosa (NOM) from five normal control subjects and 10 specimens of oral erosive or ulcerative lesions from 10 patients with erosive lichen planus (ELP) were also included. EBV-DNA was detected by PCR in 5 of the 13 (38.5%) pre-ulcerative oral aphthous lesions, two from RAU patients and three from BD patients. However, no EBV-DNA was demonstrated in five NOM specimens from normal control subjects and in 10 specimens of oral lesions from ELP patients. EBV-DNA was also demonstrated in patients' peripheral blood lymphocytes and/or plasma, suggesting that the lymphocytes may be the reservoir of latent EBV infection and there is EBV shedding in the plasma. EBV-DNA was detected by ISH in only one PCR-positive case; the reaction product was found to deposit on the nuclei of some of the epithelial cells and lymphocytes. By immunohistochemistry, expression of Epstein-Barr nuclear antigen and EBV/C3d receptors was also noted in some of the epithelial cells and lymphocytes in this ISH-positive case. Therefore, we suggest that the epithelial cells of pre-ulcerative oral aphthous lesions may be infected by EBV through EBV-infected lymphocytes; also, the cytotoxic T lymphocyte-induced lysis of the EBV-infected epithelial cells, but not the virus-induced cytolysis, may be the main mechanism causing oral ulcer formation. Our data provide preliminary evidence for an association of EBV with pre-ulcerative oral aphthous lesions in RAU and BD patients.     

Epstein-Barr virus protein LMP2A regulates reactivation from latency by negatively regulating tyrosine kinases involved in sIg-mediated signal transduction

Like other herpesviruses, Epstein-Barr Virus (EBV) persists in its host through an ability to establish latent infection with episodic reactivations. In latent infection EBV expresses an integral membrane protein LMP2A that regulates reactivation from latency. LMP2A is constitutively tyrosine phosphorylated and is associated with lyn and syk tyrosine kinases. The activity of lyn is substantially reduced. In EBV-infected cells in which LMP2A is expressed, crosslinking of sIg fails to trigger the protein tyrosine kinase signal cascade, tyrosine phosphorylation of cell proteins does not change, second messengers are not generated, and lytic EBV infection is not induced. In contrast, crosslinking of sIg on cells infected with EBV recombinants with null mutations in LMP2A results in transient tyrosine phosphorylation of lyn, syk, phospholipase C gamma 2 and phosphatidylinositol-3' kinase, transiently increased intracellular free calcium, and reactivation of lytic EBV infection. These studies describe a novel molecular regulator of herpesvirus latency and focus attention on the importance of transmembrane signal transduction in herpes virus reactivation from latency. They support the working hypothesis that the identification of ligand-receptor interactions that can result in the induction of reactivation will provide an important inroad toward the delineation of the molecular mechanism, which govern herpesvirus reactivation from latency.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

Epirubicin in hepatocellular carcinoma: pharmacokinetics and clinical activity

BACKGROUND: Severe combined immunodeficient (SCID) mice develop Epstein-Barr virus (EBV) containing human lymphoproliferative disease (LPD) tumors when reconstituted with human peripheral blood leukocytes (PBLs) from EBV-seropositive donors, but LPD tumors do not develop in the presence of immunosuppressive agents, such as cyclosporine A or corticosteroids. METHODS: Therefore, LPD development in SCID mice was used as a model to explore the relationship among B cells, T cells, and EBV in vivo. SCID mice were engrafted with PBLs isolated by leukapheresis from a single EBV-seropositive donor. Purified populations of CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) were isolated and engrafted into SCID mice. RESULTS: SCID mice engrafted with purified CD3+ lymphocytes (T cells) or CD19+ lymphocytes (B cells) did not develop LPD. In contrast, mice engrafted with purified B cells developed LPD if they were co-engrafted with purified T cells or if they were inoculated with infectious EBV. CONCLUSIONS: This study confirms the requirement of T cells or active EBV infection in the development of LPD in animals engrafted with B cells latently infected with EBV. A greater understanding of the cellular and viral interactions leading to transformation and malignancy may allow the development of specific interventional therapies for malignancies in the immunosuppressed host.     

Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1

Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types.